The present invention relates to methods of refolding proteins, particularly protein members of the transforming growth factor beta (xe2x80x9cTGF-xcex2xe2x80x9d) superfamily of proteins, such as the bone morphogenetic proteins (xe2x80x9cBMPsxe2x80x9d). These methods are particularly useful in improved processes for preparation of biologically active dimeric recombinant bone morphogenetic proteins produced in insoluble form from bacterial cell cultures.
A number of proteins referred to in the art as bone morphogenetic proteins have recently been identified which are able to induce bone or cartilage formation when implanted into mammals. For example, Wang et al. in U.S. Pat. No. 5,013,649, incorporated herein by reference, describe the DNA sequences encoding bovine and human bone morphogenetic proteins 2A (now bone morphogenetic protein-2) and 2B (now bone morphogenetic protein 4); the corresponding proteins encoded by those DNA sequences, and processes for recombinant production of the BMP-2A (now BMP-2) and BMP-2B (now BMP-4) proteins. These proteins are expected to have broad medical applicability in treatment of bone and cartilage injuries and disorders in mammals. In order to fulfill the expected medical need for these bone morphogenetic proteins, large quantities of biologically active protein will be needed.
Recombinant production of the bone morphogenetic proteins is possible both in eukaryotic and prokaryotic cell culture systems. A common occurrence in recombinant production of heterologous proteins in prokaryotic cells, such as bacteria, is the formation of insoluble intracellular precipitates known as inclusion bodies. While the bacteria are generally able to transcribe and to translate DNA sequences encoding heterologous proteins correctly, these prokaryotic cells are unable to fold some heterologous proteins sufficiently correctly to allow for their production in a soluble form. This is particularly true of prokaryotic expression of proteins of eukaryotic origin, such as the bone morphogenetic proteins. Formation of incorrectly folded heterologous proteins has to some extent limited the commercial utility of bacterial fermentation to produce recombinant mammalian proteins. When produced in bacteria, the recombinant bone morphogenetic proteins are often similarly found in inclusion bodies in an aggregated, biologically inactive form.
Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known. These methods generally involve solubilizing the protein from the inclusion bodies by denaturing the protein using acids or a chaotropic agent. Subsequently, protein is diluted into a refolding buffer that supports renaturation to a biologically active form. When cysteine residues are present in the primary amino acid sequence of the protein, it is often necessary to accomplish the refolding in an environment which allows correct formation of disulfide bonds (a redox system). General methods of refolding are disclosed in Kohno, Meth. Enzym., 185:187-195 (1990).
EP433225 describes a method for refolding transforming growth factor xcex2 (TGF-xcex2)-like proteins which employs, in addition to a chaotropic agent and a redox system, a solubilizing agent in the form of a detergent. EP433225 predicts that the methods disclosed therein are generally applicable for refolding xe2x80x9cTGF-xcex2-like proteinsxe2x80x9d, based on the degree of homology between members of the TGF-xcex2 family. However, the present inventors have found that the methods disclosed in EP433225 produce undesirably low yields of correctly folded, biologically active dimeric protein when applied to numerous bacterially produced BMPs. In addition, the methods disclosed employ 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and other expensive compounds as the solubilizing agent.
Non-detergent sulfobetaines have been used in attempts to renature hen egg white lysozyme (HEWL) and xcex2-D-galactosidase. However, these attempts have not been very effective and have practical drawbacks, such as yield. For example, certain sulfobetaines reduced the yield of xcex2-D-galactosidase by a factor of 100-fold. Goldberg et al., Folding Design., 1:21-27 (1996). Accordingly, these molecules have not been shown to be broadly effective as refolding agents, particularly for use in refolding multimeric proteins such as TGF-xcex2 proteins.
It has been found, unexpectedly, that the dimeric proteins of the TGF-xcex2 superfamily, and particularly, bone morphogenetic proteins (BMPs), can be efficiently produced and refolded from bacterial cultures, such as E. coli, using methods which employ as refolding agents non-detergent nitrogen-containing compounds for the renaturation and correct refolding of dimeric protein. Among the compounds useful in the present invention are members of the non-detergent zwitterions, pyridines, pyrroles and acid substituted aminocyclohexanes.
Accordingly, in one embodiment, the invention comprises methods of producing properly refolded proteins of the TGF-xcex2 superfamily from bacterial cell cultures using a non-detergent compound as a reagent in the method. The bacterial cell culture is preferably E. coli, but may be another bacterial or prokaryotic cell culture type. The protein may be any protein from the TGF-xcex2 superfamily, and is preferably a member of the BMP family, or the growth and differentiation factors (xe2x80x9cGDFsxe2x80x9d), as well as MP52 and other proteins as described further herein. The non-detergent compound may be nitrogen-containing and/or zwitterionic, and preferably includes an aromatic or aliphatic ring, is preferably nitrogen containing, and is preferably substituted with a substituent which includes an electrophilic or electron accepting end group, such as a carboxyl or sulfhydryl group. Other end groups which may be useful in the present invention include amide groups. The non-detergent compound is preferably selected from the group consisting of sulfobetaines, pyridines, pyrroles and aminocyclohexanes.
The non-detergent zwitterions useful in the present methods include sulfobetaines and pyridinium propanesulfonates, such as 3-(1-pyridinio)-1-propanesulfonate (xe2x80x9c3-1-PPSxe2x80x9d). Pyridine compounds useful in the present invention are preferably acid or amide substituted, and include pyridine 3-sulfonic acid, pyridine-2 carboxylic acid [also known as nicotinic acid or niacin or Vitamin B], picolinic acid, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, isonicotinic acid and nicotinamide. Pyrrole compounds which are useful in the present invention include the pyrrole analog of the above pyridine compounds. For example, pyrrole-2 carboxylic acid, the pyrrole analog of nicotinic acid, is effective in the methods of the present invention. Other non-detergent zwitterionic compounds useful in the present invention are compounds with a nitrogen containing aromatic ring, further containing an electron accepting substituent group, such as N-methyl-N-piperidine propane sulfonic acid, trigonelline hydrochloride, and 1-carboxymethyl pyridinium chloride.
Unlike pyridines and pyrroles, acid substituted aminocyclohexane compounds which are useful in the present invention contain an aliphatic ring with an amine substituent with an electron accepting group, such as a carboxyl or sulfhydryl group. For example, 2-aminocyclohexane carboxylic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-cylclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) and 2-(cylcohexylamino)ethanesulfonic acid (CHES) are each effective in the methods of the present invention.
The methods of the present invention are further advantageous in that many of these compounds are relatively inexpensive and commercially available. For example, 3-1-PPS is commercially available from Fluka Chemical Company, while Vitamin B is widely manufactured as a dietary supplement and food additive.
Among the proteins which may be produced recombinantly using the methods of the present invention are: BMP-2, BMP-3, BMP4, BMP-5, BMP-6 and BMP-7, disclosed for example in U.S. Pat. Nos. 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP-10, disclosed in PCT application WO94/26893; BMP-11, disclosed in PCT application WO94/26892, BMP-12 or BMP-13, disclosed in PCT application WO 95/16035, or BMP-15, disclosed in U.S. Pat. No. 5,635,372. Other proteins of the TGF-xcex2 superfamily which may be produced by the methods of the present invention include Vgr-2, disclosed in Jones et al., Mol. Endocrinol., 6:1961-1968 (1992); BIP, disclosed in WO94/01557; HP00269, disclosed in JP Publication number 7-250688; and MP52, disclosed in PCT application WO93/16099, and any of the growth and differentiation factors [GDFs], including those described in PCT applications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. The methods of the present invention may be used to produce commercial scale quantities of BMP homodimers or heterodimers from bacteria and refolded into biologically active dimeric molecules. Production of heterodimers of BMPs is described, for example, in WO93/19229. The disclosures of all of the above applications are hereby incorporated by reference.
Any bacterial species may be used to generate recombinant BMP for refolding in the method of the invention. Preferably, Bacillus subtilis, Pseudomonas or Escherichia coli is used to produce inclusion bodies containing BMP. Most preferably, Escherichia coli is used to produce inclusion bodies containing BMP for refolding in the method of the invention. Any strain of E. coli may be used to produce BMP for refolding in the method of the invention, so long as that strain is capable of expression of heterologous proteins. One preferred strain, E. coli strain G1724 (A.T.C.C. accession number 55151) or GI774 [without thyA] may be used to produce BMP for refolding in the method of the invention.
The methods of the present invention may be used to produce BMPs in bacteria using known methods. It may be necessary to modify the N-terminal sequences of the BMP in order to optimize bacterial expression. For example, because cleavage of the bond between formyl-methionine and glutamine is inefficient in E. coli, the N-terminus of the native mature MBP-2 protein (Met-gln-ala-lys; SEQ ID NO: 1) is modified by deletion of the glutamine residue to yield an N-terminus more suitable for BMP-2 production in E. coli (Met-ala-lys-his; SEQ ID NO: 2). Other bacterial species may require analogous modifications to optimize the yield of the mutant BMP obtained therefrom. Such modifications are well within the level of ordinary skill in the art.
The modified or unmodified nucleotide sequence which encode BMPs may be inserted into a plasmid suitable for transformation and expression of those heterologous proteins in bacteria. Any bacterial expression plasmid may be used, so long as it is capable of directing the expression of a heterologous protein such as BMP in the bacteria chosen. Acceptable species of bacteria include B. subtilis, species of Pseudomonas, and E. coli. Suitable expression plasmids for each of these species are known in the art. For production of BMP in bacteria, a suitable vector is described in Taniguchi et al., Proc. Natl. Acad. Sci.77:5230-5233 (1980).
The bacterial expression plasmid may be transformed into a competent bacterial cell using known methods. Transformants are selected for growth on medium containing an appropriate drug when drug resistance is used as the selective pressure, or for growth on medium which is deficient in an appropriate nutrient when auxotrophy is used as the selective pressure. Expression of the heterologous protein may be optimized using known methods. The BMP thus obtained will be present in insoluble, refractile inclusion bodies which may be found in pellets of disrupted and centrifuged cells.
The inclusion bodies thus obtained may be solubilized using a denaturant such as guanidine hydrochloride or by acidification with an acid such as acetic acid or formic acid. If solubilized using a denaturant, a reducing agent such as $-mercaptoethanol, glutathione, or dithiothreitol is added with the denaturant. If the protein is solubilized by acidification, it must be reduced prior to acidification.
Prior to refolding, the solubilized heterologous protein may be further purified using known chromatographic methods such as size exclusion chromatography or reverse phase high performance liquid chromatography. The solution containing the BMP may then be reduced in volume or vacuum desiccated to remove chromatography buffer and redissolved in medium. Alternatively, reduced soluble protein may be renatured by diluting into refolding medium. For example, suitable media may include the following:
(a) 50 mM Tris;
(b) 1.0 M NaCl;
(c) 0.7M 2-(cyclohexylamino)ethanesulfonic acid (CHES) or 1.0 M 3-(1-pyridinio)-propanesulfonate (3-1-PPS) or 0.4 M pyrrole-2 carboxylic acid or 0.70 M nicotinic acid;
(d) 5 mM EDTA;
(e) 2 mM gluatathione (reduced);
(f) 1 mM glutathione (oxidized);
(g) at pH of approximately 8.5
Other media may be suitable for renaturation, including media containing low levels of the chaotrope (e.g., guanidine hydrochloride) or the salt of the acid (e.g. acetate) used to solubilize the BMP inclusion bodies. Refolding is typically conducted at a BMP concentration of 1 to 100 xcexcg/ml protein. Higher concentrations of protein may be refolded in accordance with the invention, for example up to about 1 mg/ml, but precipitates or aggregates may be present above protein concentrations of 100 xcexcg/ml and the yield of active BMP homodimer or heterodimer may be decreased accordingly.
For production of heterodimers, the above procedure is performed utilizing equal amounts of two plasmids, each containing a coding sequence for a distinct BMP (e.g., pALBP2, encoding BMP-2 and pALBPX encoding BMP-X, where X is a BMP other than BMP-2). The plasmids are cultured separately, and the resulting inclusion bodies are solubilized and refolded in accordance with the methods described herein. The refolded protein monomers are mixed together in equivalent ratios and treated as described in the paragraph above. The resulting dimeric proteins are observed to include homodimers of BMP-2, as well as heterodimers of BMP-2/X. These species may be separated out from each other through procedures known in the art.
In order to refold the proteins, the following conditions and media may be used: about 10 mM to about 100 mM Tris or other suitable buffer, preferably about 50 MM Tris, about 0.1 to about 4.0 M NaCl or other suitable salt, preferably about 1.0 M NaCl, about 0.05 to about 2.0 M refolding agent [non-detergent zwitterion, sulfobetaine, pyridine, pyrrole, or aminocyclohexane], preferably about 0.7M refolding agent, about 1 mM to about 10 mM EDTA or other suitable metal ion chelating reagent, preferably about 5 mM EDTA, a suitable redox system, such as glutathione, preferably at a ratio of about 1:10 to about 10:1 reductant to oxidant, at pH of about 7 to about 11, preferably about 8.5.
Because BMPs are disulfide bonded dimers in their active state, it is useful to include a redox system which allows formation of thiol/disulfide bonds in the method of the invention. Several such redox systems are known. For example the oxidized and reduced forms of glutathione, dithiothreitol, xcex2-mercaptoethanol, xcex2-mercaptomethanol, cystine and cystamine may be used as redox systems at ratios of reductant to oxidant of about 1:10 to about 10:1. When the glutathione redox system is used, the ratio of reduced glutathione to oxidized glutathione is preferably 1 to 10; more preferably 1 to 1; and most preferably 2 to 1 of reduced form to oxidized form.
In addition to the refolding agent, the method of the invention may employ a salt moiety. The salt moiety is preferably NaCl, preferably at a concentration of about 0.1M to about 2.0M, preferably about 1.0M. It may be preferable to vary the sodium chloride concentration as the concentration of refolding agent varies.
The pH of the refolding reaction of the present invention is preferably from about 7 to about 11; more preferably from about 8 to about 10 and most preferably about 8.5.
Preferably, the refolding reaction of the invention is performed at a temperature range from about 4xc2x0 C. to about 37xc2x0 C. More preferably, the refolding reaction is performed at 20xc2x0 C. The refolding reactions of the present invention are allowed to proceed to completion between 8 and 120 hours and most preferably 96 hours.
The extent of refolding of bone morphogenetic proteins obtained is monitored by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) under non-reduced and reduced conditions. For example, the BMP4 homodimer will appear as a band of about 30 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; and the BMP-4 monomer appears as a band of about 13 kD under reduced conditions. The BMP-2/5 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-5 monomer appears as a band of about 15 kD under reduced conditions. The BMP-2/6 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-6 monomer appears as a band of about 15 kD under reduced conditions. The BMP-2/7 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-7 monomer appears as a band of about 15 kD under reduced conditions.
Without limiting the invention to a particular theory or mode of action, a unifying concept for the compounds which are useful in the present invention appears to be molecules which contain a combination of two domains, usually nitrogen-containing molecules. First, the molecule should contain a hydrophobic domain, for example, an aromatic ring such as a pyridine or pyrrole ring. Alternatively, it may be a non-aromatic ring such as cyclohexane or a non-aromatic nitrogen containing ring in which nitrogen is in the form of a quaternary amine, such as N-methyl-N-piperidine propane sulfonic acid (also known as 1-methyl-1-sulfonylpropyl piperidine). Second, the molecule should contain a substituent domain(s) which provides either zwitterionic or anionic attributes to the molecule. In a preferred embodiment, the substituent domain renders the molecule zwitterionic. In addition, the hydrophobic and substituent domains of the molecule should preferably be separated so as to present distinct regions. In a preferred embodiment, the electron accepting end group is no more than four carbons removed from the substituted aromatic or aliphatic nitrogen containing ring, more preferably no more than three carbons removed from the substituted aromatic or aliphatic nitrogen containing ring.
Accordingly, the compounds useful as refolding agents in the present methods include non-detergent zwitterionic compounds. For purposes of the present invention, it should be recognized that certain compounds may be xe2x80x9czwitterionicxe2x80x9d at selected pH ranges, but not at others. Such compounds are preferably useful in the present invention at a pH in which the compound is zwitterionic. Preferred compounds in this group include non-detergent zwitterions, such as sulfobetaines, including certain pyridines and pyrroles. Substituted aliphatic nitrogen containing rings may also be useful when nitrogen is in the form of a quaternary amine. In one preferred embodiment, the non-detergent sulfobetaine zwitterion 3-(1-pyridinio)-1-propanesulfonate (xe2x80x9c3-1-PPSxe2x80x9d), which is commercially available from Fluka Chemical Company, is useful in the present invention.
In addition, compounds comprised of an aliphatic ring such as cyclohexane substituted with an amine group and an electron accepting substituent such as carboxyl or sulfhydryl group are also effective. This class of compounds may also be referred to as acid substituted aminocyclohexanes. Included in this group of compounds are some common biological buffers, or AGood=s buffers@, including 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO). Another example of a preferred acid substituted aminocyclohexane useful in the present invention is 2-aminocyclohexane carboxylic acid.
Alternatively, compounds useful in the present methods include compounds with substituted aromatic nitrogen containing rings such as pyridines and pyrroles substituted with electron accepting groups, preferably acid groups such as carboxyl or sulfhydryl groups, or amide groups. In a preferred embodiment, the pyridine nicotinic acid, or vitamin B, is useful in the present invention. Other heterocyclic compounds, for example purines, diazines, pyrazoles and imidazoles substituted with electron accepting substituents, preferably acid groups such as carboxyl or sulfhydryl groups, or amide groups, may also be useful in the present invention, as well as derivatives of each of these compounds.
Accordingly, in one embodiment, the invention comprises methods of expressing properly refolded proteins of the TGF-xcex2 superfamily from bacterial cell cultures using one of the above refolding compounds, as described more fully below, as a reagent in the method. The bacterial cell culture is preferably E. coli, but may be another bacterial or prokaryotic cell culture type. The protein may be any protein from the TGF-xcex2 superfamily, and is preferably a member of the BMP family, or the growth and differentiation factors (xe2x80x9cGDFsxe2x80x9d), as well as MP52 and other proteins as described further herein. The refolding compound is preferably selected from the group consisting of non-detergent zwitterions, including non-detergent sulfobetaines, substituted pyridines, substituted pyrroles and acid substituted aminocyclohexanes. The refolding compound preferably comprises a hydrophobic domain, for example, an aromatic or aliphatic ring, and preferably further comprises a substituent domain, for example, an electron accepting end group, preferably an acid group such as a carboxyl or sulfhydryl group. Other substituent domains which may be useful in the present invention include amide groups. The non-detergent sulfobetaines useful in the present methods include pyridinium propanesulfonates, such as 3-(1-pyridinio)-1-propanesulfonate (xe2x80x9c3-1-PPSxe2x80x9d). Substituted pyridine compounds useful in the present invention include pyridine 3-sulfonic acid, pyridine-2 carboxylic acid [also known as nicotinic acid, niacin or Vitamin B], picolinic acid, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, isonicotinic acid and nicotinamide. Substituted pyrrole compounds which are useful in the present invention include the pyrrole analog of the above pyridine compounds. For example, pyrrole-2 carboxylic acid, pyrrole 3-sulfonic acid, 3-pyrrole acetic acid hydrochloride, 2-pyrrole acetic acid hydrochloride 2-pyrrole ethane sulfonic acid, 3-pyrrolehydroxymethane sulfonic acid. Among the substituted aminocyclohexanes which are useful in the present invention are 2-aminocyclohexanecarboxylic acid, and Good""s Buffers, such as CHES, CAPS and CAPSO. Illustrated below are some compounds which are useful in the present invention.
Non-Detergent Zwitterions, Subsituted Pyridines and Nicotinic Acid Derivatives 
Substituted Pyrroles 
Unlike the substituted pyridines and pyrroles, substituted aminocyclohexanes which are useful in the present invention contain an aliphatic ring with an amine subsituent with an electron accepting end group, such as a carboxyl or sulfhydryl group. For example, CHES, CAPS and CAPSO are each effective in the methods of the present invention. Illustrated below are various compounds which are useful.
Substituted Aminocyclohexanes 
The in vitro biological activity of the refolded bone morphogenetic proteins may be monitored by the W-20 assay as set forth in the examples. Use of the W-20-17 bone marrow stromal cells as an indicator cell line is based upon the conversion of these cells to osteoblast-like cells after treatment with BMP [Thies et al., Journal of Bone and Mineral Research 5(2): 305 (1990); and Thies et al., Endocrinology 130: 1318-1324 (1992)]. W-20-17 cells are a clonal bone marrow stromal cell line derived from adult mice by researchers in the laboratory of Dr. D. Nathan, Children""s Hospital, Boston, Mass. Treatment of W-20-17 cells with BMP results in (1) increased alkaline phosphatase production, (2) induction of parathyroid hormone stimulated cAMP, and (3) induction of osteocalcin synthesis by the cells. While (1) and (2) represent characteristics associated with the osteoblast phenotype, the ability to synthesize osteocalcin is a phenotypic property only displayed by mature osteoblasts. Furthermore, to date the conversion of W-20-17 stromal cells to osteoblast-like cells has been observed only upon treatment with bone morphogenetic proteins.